The phenotypic screens described in the accompanying Discovery Pipeline proposal do not directly identify the small-molecule target, yet target identification is vitally important to follow-up, medicinal chemistry efforts. Target identification, including both the therapeutic target and "off targets" that can result in unwanted "side effects", enables optimization of a compound's selectivity and reduction in the potential for side effects of a resultant drug. Target discovery and validation as it is currently practiced is a significant bottleneck to drug discovery. This proposal offers a solution to the target identification problem through the systematic use of three distinct and interdisciplinary approaches. Proteomic, genetic and computational methodologies have been developed at the Broad Institute and will be implemented as a critical follow-up to screening results. Specifically, we will: 1) develop a systematic, proteomic approach to identify targets that underlie smallmolecule screening discoveries; 2) combine small-molecule and RNAi methods to identify or to validate targets that underlie small-molecule screening discoveries; and 3) develop computational methods based on gene expression, small-molecule screening, and proteomic and RNAi data to identify pathways and targets that underlie small-molecule screening discoveries. The problem of target identification is one that may be solved not by a single 'killer' technology; rather, we envision the analytical integration of multiple, complementary approaches. Each of these is dependent upon a focused, interdisciplinary effort to a problem that is unlikely to be solvable with the expertise of one discipline alone. We are proposing to develop small-molecule drugs to alleviate unmet medical needs. In order to maximize a drug's potency and minimize a drug's unwanted side-effects, we need to understand all the proteins ("targets") that a drug candidate will interact with. This proposal seeks to use a combination of cutting-edge scientific approaches available at the Broad Institute to address the difficult problem of target identification.